Project Summary/Abstract The project outlined in this proposal will allow the characterization of vertebrate multi-lineage hematopoietic progenitor cells (HPCs) that have the capability of generating mature erythroid, myeloid, and thrombocytic cells in adult organisms. These cells and their lineage-specific differentiation are essential; perturbations in these processes lead to serious diseases in humans including anemia, neutropenia, and thrombocytopenia. While understanding the genetic regulation of these cells is critical for treating and curing human hematopoietic disease, very little is known about the molecular pathways that control their generation, proliferation, and especially their differentiation and maturation. The objective of this project is to understand these normal developmental processes that are dysregulated during disease so that they can be manipulated to cure and treat a multitude of hematopathologies. To identify novel pathways involved in these cells, Danio rerio (zebrafish) will be employed. Zebrafish are an excellent vertebrate system to study blood development; they are optically transparent, fecund, and genetically amenable, explaining their utility in studying blood development and disorders. Importantly, zebrafish have served as a model organism to study human hematopoietic disease, as their blood system is nearly identical to mouse and human. Many forward genetic screens have been performed in zebrafish and successfully identified essential genes in primitive red blood cell development, mainly because perturbations in red blood cell development are easily visually identified. However, mutations in HPCs, the cells directly related to hematopoietic disease, were likely missed in these screens. Over the past seven years, sensitive in vitro assays, similar to the ones used to prove the existence of mammalian HPCs, have been developed that finally allow the functional study of these essential cells in the zebrafish system. This project will utilize these assays to carefully analyze zebrafish HPCs for the first time. Individual populations of HPCs will be isolated and interrogated to see what genes, micro RNAs (miRNAs), and long non- coding RNAs (lncRNAs) are enriched within them; comparing these genes to those of mammalian HPCs will allow an understanding of evolutionarily conserved pathways involved in their biology. Furthermore, forward mutagenesis screens will be performed on HPCs to determine, in an unbiased manner, genes essential for their generation and differentiation into mature blood cells. After validating the function of these genes in zebrafish and other vertebrate animals, it will finally be possible to understand the normal development of HPCs, and the molecular pathways dysregulated during hematopoietic disease. With the knowledge of molecular pathways involved in the essential, enigmatic processes of HPC formation and differentiation, treatments to common hematologic disorders can be devised and implemented.